PDBsum entry 3a8r

Calcium binding protein

PDB id: 3a8r

Contents

Protein chains

166 a.a. *

Metals

_CA ×2

Waters ×77

* Residue conservation analysis

PDB id:

3a8r

Name:

Calcium binding protein

Title:

The structure of the n-terminal regulatory domain of a plant NADPH oxidase

Structure:

Putative uncharacterized protein. Chain: a, b. Fragment: ef-hand domain, residues 138-313. Synonym: rbohb, os01g0360200 protein, respiratory burst oxidase protein b. Engineered: yes

Source:

Oryza sativa japonica group. Japanese rice. Organism_taxid: 39947. Gene: b1164g11.26, os01g0360200, osj_01746. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.40Å R-factor: 0.235 R-free: 0.266

Authors:

T.Oda,H.Hashimoto,N.Kuwabara,S.Akashi,K.Hayashi,C.Kojima,H.L.Wong, T.Kawasaki,K.Shimamoto,M.Sato,T.Shimizu

Key ref:

T.Oda et al. (2010). Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications. J Biol Chem, 285, 1435-1445. PubMed id: 19864426 DOI: 10.1074/jbc.M109.058909

Date:

07-Oct-09 Release date: 27-Oct-09

Protein chains

Q5ZAJ0  (RBOHB_ORYSJ) -  Respiratory burst oxidase homolog protein B from Oryza sativa subsp. japonica

Seq: 905 a.a.

Struc: 166 a.a.

Enzyme reactions

• Enzyme class 1: E.C.1.11.1.- - ?????
• Enzyme class 2: E.C.1.6.3.- - ?????

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1074/jbc.M109.058909

J Biol Chem 285:1435-1445 (2010)

PubMed id: 19864426

Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications.

T.Oda, H.Hashimoto, N.Kuwabara, S.Akashi, K.Hayashi, C.Kojima, H.L.Wong, T.Kawasaki, K.Shimamoto, M.Sato, T.Shimizu.

ABSTRACT

Plant NADPH oxidases (Rboh; for Respiratory Burst Oxidase Homolog) produce reactive oxygen species (ROS) that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs which regulates Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca2+ binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping of EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca2+-binding to the only one EF-hand. Structure-based in vitro pull-down assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by the swapping of the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N- and the C-terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of ROS production.

Selected figure(s)

Figure 2.
EF-hand motifs of OsRbohB-(138–313) and similar proteins. A, representation of EF-hand motifs (EF-1, EF-2, EF-like 1, and EF-like 2). Difference Fourier maps showing contour levels higher than 5 σ. Ca^2+ ion and water molecules are represented as a yellow and red spheres, respectively. Amino acid residues of EF-hand motifs at positions X to Z are shown as sticks. B, magnified view of EF-hand pairs composed of EF-1 and EF-2, and EF-like 1 and EF-like 2, respectively. Residues forming the hydrophobic cores are shown as white sticks. C, hydrophobic pockets of OsRbohB-(138–313), calcineurin B, and recoverin. Residues forming the hydrophobic pocket are shown as white sticks. The pocket of OsRbohB-(138–313) formed by swapped EF-hands and EF-hand-like motifs is occupied by an N-terminal helix (blue) protruding from the core domain. The pockets of calcineurin B and recoverin are occupied by α-helices protruding from calcineurin A and the C-terminal region (blue), respectively.

Figure 4.
Interaction between OsRbohB-(138–313) and OsRac1. In vitro pulldown assays using OsRbohB-(138–313) mutants and GST-OsRac1. Mutation and deletion sites are mapped onto the structure of OsRbohB-(138–313) shown as a ribbon model and as surface representations (two views from opposite sides). Residues that are necessary to maintain the full binding affinity are colored magenta, whereas residues exhibiting no or little effect on the binding are shown in blue.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2010, 285, 1435-1445) copyright 2010.

Figures were selected by an automated process.